Plasminogen activator as an anti-inflammatory agent

ABSTRACT

Plasminogen activator acts as an anti-inflammatory agent by inhibiting generation of superoxide anion by a mechanism that is not related to L-arginine, is not dependent on thrombolytic activity, and is not a function of oxygen free radical scavenging. Moreover, in in vivo models of inflammation, treatment with plasminogen activator reduces edema without inhibiting neutrophil infiltration in in vivo models of inflammation.

This application is the national phase of international applicationPCT/US98/01948 filed Jan. 29, 1998 under 35 U.S.C. 371 which designatedthe U.S. This application also claims the benefit of U.S. ProvisionalApplication No. 60/036,566, filed Jan. 27, 1997.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the use of plasminogen activator as ananti-inflammatory agent.

2. Description of the Related Art

Plasminogen activators play an important physiological role in theregulation of thrombolysis. This action is exploited therapeutically topromote reperfusion in conditions such as, for example, acute myocardialinfarction, pulmonary embolism, and thrombotic stroke.

An equilibrium between two opposing reactions, coagulation which formsblood clots and fibrinolysis which dissolves blood clots, maintains apatent and intact vascular endothelium. To stop blood loss from aleaking blood vessel, blood clots form a hemostatic plug at the site ofa break in the vessel wall. But if the blood clot obstructs flow througha blood vessel, the result may be, for example, a myocardial infarction,a pulmonary embolism, or a thrombotic stroke.

The interruption of flow through the blood vessel will lead to tissueischemia. In this condition, the tissue is deprived of oxygen andbecomes jeopardized, a state in which the tissue is injured but stillpotentially viable. If however the hypoxic condition is maintained for aperiod of several hours, the tissue becomes necrotic and cannot recover.It is therefore important that reperfusion, the restoration of bloodflow, be accomplished as soon as possible to minimize tissue necrosis.However, even if reperfusion is accomplished before tissue necrosisoccurs, leukocytes may become activated and infiltrate the jeopardizedtissue. Consequently, reperfusion only leads to partial recovery ofjeopardized tissue, the remainder being permanently damaged by leukocytemediated oxidative injury or other pathophysiological mechanisms(Hansen, Circulation 91:1872-1885, 1995).

In particular, patients with acute myocardial infarction havesignificantly reduced mortality when treated with a plasminogenactivator. This benefit is due to blood clot fibrinolysis and timelyopening of the infarct-related artery. However, once the infarct-relatedartery is patent, neutrophils can contribute to the reperfusion injurythat accompanies reversal of ischemia. Reperfusion of the myocardium isassociated with neutrophil activation and infiltration. The nature ofthe neutrophil-mediated injury is not fully characterized but is in partdue to the production of superoxide anion (O₂ ⁻) and/or relatedoxidative products. This principle (activation of white blood cellsrelease of toxic mediators and resultant pathophysiology in the host) iscommon to many inflammatory diseases including, but not limited to,acute respiratory distress syndrome, cystic fibrosis, asthma, arthritis,and nephritis.

A drawback of thrombolytic therapy with plasminogen activator is theincreased incidence of stroke and intracerebral hemorrhage in treatedpatients. The generation of fibrin fragments and depletion of fibrinogencaused by the exogenous plasminogen activator is a direct cause ofexcessive bleeding disorders.

Side-effects of existing anti-inflammatory agents such as steroidal(e.g., corticosteroids) and non-steroidal (e.g., aspirin, acetaminophen,ibuprofen) drugs include growth retardation, osteoperosis, gastric andrenal toxicity, and adrenal suppression. In addition, these agents canimpair the healing process due to inhibition of neutrophil infiltrationby steroidal and non-steroidal drugs. Most non-steroidal drugs are alsolimited to oral formulations.

We have now found that plasminogen activator can reduce tissue injury byacting as an anti-inflammatory agent. More generally, plasminogenactivator reduces tissue damage due to leukocyte mediated oxidativeinjury. Plasminogen activator inhibits leukocyte generation of oxygenradicals (e.g., hydroxides, peroxides, superoxides) by a mechanism thatis independent of thrombolytic activity and scavenging of oxygen freeradicals. By separating the thrombolytic and the anti-inflammatoryfunctions of plasminogen activator, the present invention provides amethod of reducing tissue damage due to oxidative injury (e.g.,reperfusion injury) while mitigating complications from excessivebleeding, such as stroke and intracerebral hemorrhage. Moreover, becausethe present invention does not inhibit neutrophil migration andinfiltration, use of plasminogen activator as an anti-inflammatory agentwill not interfere with processes mediated at least in part byneutrophils such as, for example, wound healing or tissue remodeling,which is a shortcoming of existing steroidal and non-steroidalanti-inflammatory agents.

SUMMARY OF THE INVENTION

It is an object of the invention to reduce cell and/or tissue damage dueto oxidative injury.

It is a further object of the invention to inhibit oxidant production byleukocytes.

Another object of the invention is to use plasminogen activator as ananti-inflammatory agent.

Yet another object of the invention is to treat a human or animalafflicted with an inflammatory disease.

It is an object of the invention to select and/or develop derivatives ofplasminogen activator that retain the anti-inflammatory property ofplasminogen activator.

Another object of the invention is to provide a derivative ofplasminogen activator that can be used as an anti-inflammatory agent.

Yet another object of the invention is to provide a non-thrombolyticform of plasminogen activator useful as an anti-inflammatory agent.

In one embodiment of the invention, a plasminogen activator orderivative thereof is administered to an organism, and thereby reducesoxidative injury to tissue of the organism. Preferably, the organism isafflicted with an autoimmune disease or an inflammatory disease. Theorganism may be a human or an animal. The oxidative injury may bemediated by an inflammatory cell (e.g., neutrophil, macrophage,monocyte, eosinophil, mast cell, basophil). Tissue at risk of oxidativeinjury may include, but is not limited to, any blood-prefused tissue(e.g., myocardiun, lung, brain) or any bone or joint.

In a second embodiment of the invention, a plasminogen activator orderivative thereof is applied to an inflammatory cell (e.g., neutrophil,macrophage, monocyte, eosinophil, mast cell, basophil), and therebyreduces oxidant production by the inflammatory cell.

A third embodiment of the invention is the treatment of an organismafflicted with an inflammatory disease by administering a plasminogenactivator or derivative thereof, and thereby reducing or alleviating asymptom of the disease caused by inflammation. The organism may be ahuman or an animal. Preferably, the organism is afflicted with an acuteor chronic inflammatory disease.

A fourth embodiment of the invention is the prophylactic treatment of anorganism at risk for development of an inflammatory illness byadministering a plasminogen activator or derivative thereof, and therebypreventing onset of the illness or reducing the severity of a symptom ofthe illness. The organism may be a human or an animal. Preferably, theorganism is at risk for development of an acute or chronic inflammatoryillness.

For the above embodiments of the invention, plasminogen activator may betissue-plasminogen activator (tPA), urokinase (uPA), reteplase (rPA), ora derivative thereof. Such plasminogen activators may be termed“endogenous plasminogen activators” to distinguish them fromstreptokinase. Tissue plasminogen activator and urokinase are “mammalianplasminogen activators” whereas streptokinase is produced by bacteria.For these two definitions, no distinction is made between native proteinand recombinantly produced protein (e.g., non-glycosylated). Instead,“endogenous” may mean derived from the species of the organism or theinflammatory cell being treated and “mammalian” indicates the geneencoding the plasminogen activator is derived from a mammal.

Preferred derivatives of endogenous plasminogen activator have theproperty of reduced fibrinolytic activity, binding a receptor forplasminogen activator, inhibiting oxidant production, inhibitingleukocyte activation, or a combination thereof. More preferably, thederivative of endogenous plasminogen activator binds urokinase receptorwith increased affinity, reduces production of superoxide anion by aninflammatory cell (e.g., neutrophil, macrophage, monocyte, eosinophil,mast cell, basophil), reduces release of other mediators of inflammation(e.g., arachidonate metabolites, cytokines, histamine, monokines, nitricoxide, proteases, serotonin) by an inflammatory cell or the endothelium,or a combination thereof. The 15 Kd amino-terminal fragment (ATF) (aminoacid residues 1-135) of urokinase is an example of such a derivative,binding the urokinase receptor but lacking fibrinolytic activity.

A fifth embodiment of the invention is to provide structural variants ofan endogenous plasminogen activator, to screen the structural variantsfor their ability to reduce inflammation, and to select those structuralvariants which reduce inflammation. The structural variant may beproduced recombinantly, by peptide synthesis, by protease cleavage, orby chemical modification. Preferred structural variants have theproperty of reduced fibrinolytic activity, binding a receptor forplasminogen activator, inhibiting oxidant production, inhibitingleukocyte activation, or a combination thereof. The 15 Kd amino-terminalfragment (ATF) (amino acid residues 1-135) of urokinase is an example ofsuch a preferred structural variant, binding the urokinase receptor butlacking fibrinolytic activity. More preferably, the structural variantbinds the receptor for plasminogen activator with increased affinity,reduces production of oxidant radicals and/or another marker ofleukocyte activation, reduces cellular degranulation, reduces vascularpermeability, or a combination thereof.

Endogenous plasminogen activator may be tissue-plasminogen activator(tPA), urokinase (uPA), or a derivative thereof. A plasminogenderivative with reduced fibrinolytic activity is preferred, but notnecessary. A plasminogen activator derivative that binds the plasminogenactivator receptor on leukocytes, thereby inhibiting production ofoxidants and/or other markers of leukocyte activation, is also preferredbut not necessary.

The advantages of the invention include the ability to inhibitinflammation and tissue injury using a plasminogen activator that doesnot cause excessive bleeding, and provision of a novel class ofanti-inflammatory agents that reduces tissue injury caused by leukocyteproduction of oxidants and proteases, without interfering with otherleukocyte functions such as, for example, migration and infiltration.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the effect of tPA on O₂ ⁻ production by human neutrophilsstimulated with PMA in vitro. Adding tPA concentrations of 20-100 μg/mlsignificantly (asterisk represents p<0.05) reduced neutrophil O₂ ⁻production compared to values obtained following no additions oraddition of 5 μg/ml tPA. Each value is the mean±standard error of threeor more determinations.

FIG. 2 shows the effect of L-arginine on O₂ ⁻ production by humanneutrophils in vitro. Adding 175 or 700 μg/ml of L-arginine, a componentof the tPA preparation, did not decrease (p>0.05) O₂ ⁻ production by PMAstimulated neutrophils in vitro. By comparison, adding 1400 or 3500μg/ml of L-arginine increased (asterisk represents p<0.05) O₂ ⁻production by PMA stimulated neutrophils in vitro. Each value is themean±standard error of three or more determinations.

FIG. 3 shows the effect of tPA or PPACK treated tPA on O₂ ⁻ productionby neutrophils in vitro. Compared to PMA alone, neutrophils treated withtPA or PPACK treated tPA had comparable (p>0.05) decreases in O₂ ⁻generation. Neutrophils were pretreated with tPA, PPACK, or PPACK:tPA(5:1 mole ratio) and subsequently activated by PMA. The time course ofcytochrome C reduction (O₂ ⁻ production) was monitored by changes inoptical density (mOD). Neither neutrophils alone, tPA alone, PPACKalone, or acetic acid (PPACK vehicle) altered cytochrome C reduction.The Vmax (mOD/min) or rate of cytochrome C reduction was significantlyless for both tPA+PMA, filled square (0.198±0.09), and PPACK:tPA+PMA,open triangle (0.182±0.01), when compared to PMA alone, filled diamond(0.476±0.15) with p=0.0004 and 0.006, respectively. In addition, therewas no significant difference in Vmax between the tPA+PMA andPPACK:tPA+PMA groups (p>0.05). Data are means of triplicate experiments.

FIG. 4 shows the effect of tPA on produced by xanthine oxidase in vitro.Adding increasing amounts of tPA did not decrease (p>0.05) generation byxanthine oxidase (XO) in vitro. Each value is the mean±standard error ofthree or more experiments.

FIG. 5 shows the effect of tissue plasminogen activator (tPA) oncarrageenan induced edema in rat footpad. Open square, saline alone;filled square, carrageenan alone; filled triangle, 12 mg/KgtPA+carrageenan; filled circle, 6 mg/Kg tPA+carrageenan; open circle, 3mg/Kg tPA+carrageenan. Edema index reflects changes in hind paw volumeat different times after plantar carrageenan or saline administration.Data represent the means (±SEM) for ten experiments. Asterisk representsp≦0.05 tPA vs. carrageenan alone.

FIG. 6 shows the effect of streptokinase (SK) on carrageenan inducededema in rat footpad. Open square, saline alone; filled square,carrageenan alone; filled triangle, 40,000 U/Kg SK+carrageenan; filledcircle, 20,000 U/Kg SK+carrageenan; open circle, 10,000 U/KgSK+carrageenan. Edema index reflects changes in hind paw volume atdifferent times after plantar carrageenan or saline administration. Datarepresent the means (±SEM) for ten experiments. Asterisk representsp≦0.05 for SK vs. carrageenan alone.

FIG. 7 shows modulation of IL-1 induced lung leak. Data are presented asthe means±SEM. Asterisk represents a p value of <0.05 vs. IL-1 controlgroup. Lung leak induced by L-arginine was not significantly differentthat that induced by saline alone.

FIG. 8 shows tPA induced inhibition of oxidant production by a ratalveolar macrophage line. Cells were pretreated with tPA (100 μg/ml) orvehicle, and subsequently exposed to phorbol ester (PMA, 1.25 μg/ml),zymosan (ZMA, 60 μg/ml), or opsonized zymosan (opZMA, 60 μg/ml). Largeopen circle, control; large closed circle, tPA; open square, PMA; closedsquare, PMA+tPA; open triangle, ZMA; closed triangle, ZMA+tPA; smallopen circle, opZMA; and small closed circle, opZMA+tPA. Data representmean values from triplicate estimations.

FIG. 9 shows tPA alone significantly reduced the rate of apoptosis andthe percent apoptotic cells at 24 hours. Open circle, cells alone;closed circle, tPA alone; open square, PMA alone; closed square, fMLPalone; open triangle, tPA+PMA; and closed triangle, tPA+fMLP.

DETAILED DESCRIPTION OF THE INVENTION

Blood coagulation is a complex process consisting of interactions ofvarious blood components which eventually gives rise to a fibrinnetwork, or blood clot. Degradation of the fibrin network can beaccomplished by activationof the zymogen plasminogen to plasmin. Plasminis a serine protease which actsdirectly to degrade the fibrin networkand thereby regulate the coagulation process. Conversion of plasminogeninto plasmin is normally catalyzed in vivo by tissue plasminogenactivator (tPA), a fibrin-specific serine protease which is believed tobe the physiological vascular activator of plasminogen. Urokinaseplasminogen activator (uPA) is another member of the class ofplasminogen activators characterized as serine proteases. tPA and uPAare functionally and immunologically distinguishable (reviewed in“Thrombolytic Agents” by Collen et at. and “Principles of ThrombolyticTherapy in Myocardial Infarction” by Sutton et al., in Singh et al.,Eds., Cardiovascular Pharmacology and Therapeutics).

Tissue Plasminogen Activator

Human tPA is a multidomain serine protease secreted by vascularendothelial cells. Five distinct structural domains make up the 527amino acids of the active human tPA protein. The DNA and amino acidsequences of human tPA was described by Pennica et al. (Nature301:214-221, 1983). The numbering system employed by Pennica et al. isused herein. The gene encoding tPA is comprised of 12 exons split byintrons (Ny et al., Proc. Natl. Acad. Sci. USA 81:5355-5359, 1984).These introns correspond, in part, to the junction of the domainsdescribed below.

The amino-terminal portion of the molecule contains a disulfide-linkedloop referred to as the Finger (F) domain (amino acid residues 1-43).This domain is highly homologous to the Finger domain of fibronectin andprovides this molecule with fibrin-binding properties. The seconddomain, called the Epidermal Growth Factor-like (EGF) domain (amino acidresidues 44-91), is highly homologous with epidermal growth factor.Similar EGF domains occur in serine proteases such as protein C,clotting factors VII, IX and X, and urokinase. The third and fourthdomains are highly disulfide-linked structures referred to as KringlesK1 and K2 (amino acid residues 92-173 and 180-261, respectively).Similar kringle structures are present in plasma proteins such asprothrombin, plasminogen, and urokinase and are also believed to beimportant in binding fibrin. The fifth domain, located at thecarboxy-terminus, is the Serine Protease (SP) domain (amino acidresidues 262-527) The SP domain is homologous to similar domains inplasma clotting serine proteases, urokinase, and trypsin, and containsthe active site for the fibrin-specific serine protease activity (aminoacid residues His³²², Asp³⁷¹, and Ser⁴⁷⁸).

The precursor form of tPA additionally comprises a pre-region followeddownstream by a pro-region, which are collectively referred to as the“pre-pro” region. The pre-region contains a signal peptide which isimportant for protein secretion of tPA by cells. The pre-sequence isbelieved responsible for secretion of tPA into the lumen of theendoplasmic reticulum, a necessary step in extracellular secretion. Thepro-sequence is believed to be cleaved from the tPA molecule followingtransport from the endoplasmic reticulum to the Golgi complex.

tPA usually circulates as a single polypeptide chain of M_(r)approximately 72 Kd, which is converted to a two-chain form by cleavageof the peptide bond cleavage between Arg²⁷⁵ and Ile²⁷⁶. The heavy chainof tPA (two variants of M_(r) 40 Kd and 37 Kd) is derived from theamino-terminus, while the light chain (M_(r) 33 Kd) is derived from thecarboxy-terminal end of the tPA molecule. This cleavage is catalyzed bytrypsin or plasmin, and is accompanied by an increase in activity, asmeasured using synthetic substrates, and by an increase in fibrinolyticactivity. Single-chain tPA becomes active upon binding to fibrin,probably due to a conformational change in the activator induced bybinding to fibrin. Recombinant tPA (i.e., alteplase) is provided as aone-chain polypeptide that is cleaved in vivo to an active two-chainpolypeptide.

The gene and protein for tPA, methods of assaying for various propertiesof tPA, methods of making derivatives and structural variants of tPA,methods of expressing and purifying tPA, and other information aredescribed in U.S. Pat. Nos. 4,766,075, 4,963,357, 5,094,953, 5,106,741,5,108,901, 5,149,533, 5,156,969, 5,232,847, 5,242,688, 5,246,850,5,270,198, 5,275,946, 5,486,471, and 5,556,621, incorporated herein byreference.

Urokinase and Receptor for Plasminogen Activator

uPA is released from many types of cultured cells as a single-chainproenzyme with little or no plasminogen activating capacity. By limitedproteolysis with catalytic amounts of plasmin, this proenzyme can beconverted to its active two-chain counterpart. The proenzyme nature ofsingle-chain uPA is also reflected in the finding that it hasessentially no amidolytic activity with synthetic substrates, and thatit has little or no reactivity with macromolecular inhibitors andsynthetic inhibitors.

In the intact organism, pro-uPA is the predominant form of uPA inintracellular stores, and it also constitutes a sizable fraction of theuPA in extracellular fluids. Extracellular activation of pro-uPA istherefore be a crucial step in the physiological regulation of the uPApathway of plasminogen activation. The plasmin-catalyzed activation ofpro-uPA provides a positive feedback mechanism that accelerates andamplifies the effect of activation of a small amount of pro-uPA.However, plasmin-resistant single-chain derivatives of urokinase do havefibrinolytic activity (Gurewich et al., J. Clin. Invest. 82:1956-1962,1988).

The urokinase molecule exists is several biologically active forms: highmolecular weight (54 Kd) and low molecular weight (33 Kd), each composedof single-chain or two-chain material; the low molecular weight form isderived from the high molecular weight form by enzymatic cleavage of thepeptide bond between Lys¹⁵⁸ and Ile¹⁵⁹. Human uPA is encoded by a genewith 11 exons and has an N-terminal Epidermal Growth Factor-like (EGF)domain implicated in receptor binding (mainly exon IV) and a singleKringle domain (exons V and VI), followed by a Serine Protease (SP)domain (exons VII-XI) for a polypeptide of 411 amino acids (Riccio etal., Nucl. Acids Res. 13:2759-2771, 1985). The active site comprisesamino acid residues His₂₀₄, Asp²⁵⁵, and Ser³⁵⁶. The numbering system ofRiccio et al. is used herein.

The cellular receptor for uPA (uPAR) is found on cells such asleukocytes (Felez et al., Blood 78:2318-2327, 1991; Min et al., J.Immunol. 148:3636-3642, 1992; Plesner et al., Am. J. Clin. Path.102:835-841, 1994). Human uPAR has been characterized and cloned (Roldanet al., EMBO J. 9:467-474, 1990) as a 55-60 Kd glycoprotein. Thereceptor binds active 54 Kd uPA, its single polypeptide chain proenzyme,pro-uPA as well as 54 Kd uPA inhibited by the active site reagent DFP,but shows no binding of the 33 Kd form of active uPA. Thus, binding tothe receptor does not require the catalytic site of uPA, and inagreement with these findings, the binding determinant of uPA has beenidentified in the amino-terminal part of the enzyme, in a region whichin the primary structure is remote from the catalytic site. The receptorbinding domain is located in the 15 Kd amino-terminal fragment (ATF)(amino acid residues 1-135) of uPA (Stoppelli et al., Proc. Natl. Acad.Sci. USA 82:4939-4943, 1985), more precisely within the EGF domain(Appella et al., J. Biol. Chem. 262:4437-4440, 1987). The uPA amino acidresidues which appear to be critical for receptor binding are locatedwithin amino acid residues 12-32 (Appella et al., ibid.; Magdolen etal., Eur. J. Biochem. 237:743-751, 1996); the peptides described byAppella et al. and Magdolen et al. may be used to demonstrate thecorrelation between receptor binding and the anti-inflammatory activityof plasminogen activator.

The gene and protein for uPA and uPAR, methods of assaying for variousproperties of uPA and uPAR (e.g., ligand-receptor binding), methods ofmaking derivatives and structural variants of uPA and uPAR, methods ofexpressing and purifying uPA and uPAR, and other information aredescribed in U.S. Pat. Nos. 4,326,033, 4,370,417, 5,112,755, 5,175,105,5,219,569, 5,240,845, 5,472,692, 5,519,120, 5,550,213, and 5,571,708incorporated herein by reference.

Proteins have been defined by means of determined DNA sequence anddeduced amino acid sequence; it will be understood that natural allelicvariation may exist within a species.

The above tPA or uPA protein may be obtained from natural sources;produced by recombinant techniques in bacteria, yeast, or mammaliancells; and/or synthesized chemically. Derivatives and structuralvariants of tPA or uPA proteins may be produced by chemical orproteolytic cleavage of the native protein, peptide synthesis, and/orrecombinant techniques. Such derivative and structural variants may begenerated by random mutagenesis; domain swapping, deletion, orduplication; and/or directed mutagenesis. Derivatives and structuralvariants of tPA or uPA proteins may contain amino acid substitutions,deletions, additions and/or replacements. The molecular interactionbetween substrate and enzyme (U.S. Pat. Nos. 5,433,940 and 5,464,820,incorporated herein by reference), or ligand and receptor (Wells,Bio/Technology 13:647-651, 1995 and U.S. Pat. No. 5,534,617,incorporated herein by reference) may also be used to design derivativesand structural variants of tPA or uPA. Protein may be fractionated andpurified by its physical and/or chemical characteristics (Janson andRyder, Protein Purification, VCH, New York, 1989; Scopes, ProteinPurification, Springer-Verlag, New York, 1993).

Non-thrombolytic forms of plasminogen activator may be produced by meanssuch as, for example, incubation with a serine protease inhibitor asdescribed in U.S. Pat. No. 5,304,482 incorporated herein by reference,formation of a complex with a plasminogen activator inhibitor (PAI),isolation of a plasminogen activator fragment after chemical orenzymatic cleavage, and/or genetic engineering. The proteolytic activityof tPA can be inhibited by PPACK. The tPA-PPACK complex retained itsability to inhibit human neutrophil O₂ ⁻ production in vitro (Stringeret al., Inflammation 21:27-34, 1997). A derivative of plasminogenactivator may be expressed by recombinant DNA technology (Goeddel, GeneExpression Technology, Academic Press, San Diego, 1990; Kriegler, GeneTransfer and Expression, Stockton Press, New York, 1990; Ausubel et al.,Current Protocols in Molecular Biology, Wiley, New York, 1996); suchderivatives may contain deletions in the serine protease (SP) domain,and/or mutations that reduce or eliminate the serine protease activityof plasminogen activator. After treatment to inactivate the proteaseactivity of plasminogen activator, the plasminogen activator derivativemay be isolated by fractionating the treated plasminogen activator andassaying for fractions containing an activity which inhibits oxidantproduction by leukocytes.

The compounds of the invention may be formulated according to knownmethods to prepare pharmaceutical compositions, whereby the plasminogenactivator or derivative thereof is combined with a pharmaceuticallyacceptable carrier vehicle. Suitable vehicles and their formulation aredescribed, for example, in Remington's Pharmaceutical Sciences by E. W.Martin. Such compositions will contain an effective amount of theplasminogen activator or derivative thereof together with a suitableamount of vehicle in order to prepare pharmaceutically acceptablecompositions suitable for administration to a human or animal.Formulations or other delivery systems (e.g., liposomes) will besuitable for oral, topical, inhalation, or parenteral administration.

Compositions of the invention may include an inhibitor of a proteasereleased during inflammation by leukocytes (e.g., cathepsin G, chymase,elastase, tryptase), an oxidant scavenger (e.g., superoxide dismutase,see for example U.S. Pat. No. 4,976,959, incorporated herein byreference), a growth factor (see for example U.S. Pat. No. 5,057,494,incorporated herein by reference) and/or an inhibitor of interleukin-1(U.S. Pat. Nos. 5,075,222, 5,359,032, 5,453,490, 5,455,330, and5,521,185, incorporated herein by reference), in addition to plasminogenactivator or a derivative thereof. Leukocyte proteases and inhibitorsare described in U.S. Pat. Nos. 5,420,110, 5,541,288, 5,455,229,5,510,333, and 5,525,623, incorporated herein by reference. Compositionsof the invention may include a protease inhibitor such as, for example,α₁-antiprotease, α₁-antitrypsin (AAT), aprotinin,3,4-dichloro-isocoumarin, diisopropyl fluorophosphate (DFP),α₂-macroglobulin, phenylmethylsulfonyl fluoride (PMSF), plasminogenactivator inhibitor (PAI), secretory leukoprotease inhibitor (SLPI),and/or urinary trypsin inhibitor (UTI), in addition to plasminogenactivator or a derivative thereof.

An effective amount of a compound of the invention may depend upon anumber of factors including, for example, the age and weight of thehuman or animal, the precise condition requiring treatment and itsseverity, and the route of administration. The precise amount willultimately be at the discretion of the attending physician orveterinarian. Thus, practice of the present invention may involve anydose, combination (with another plasminogen activator or other agents),reformulation, or delivery system (e.g., liposomes) for oral, topical,inhalation, or parenteral administration.

Intravascular infusions are normally carried out with the parenteralsolution contained within an infusion bag or bottle, or within anelectrically operated infusion syringe. The solution may be deliveredfrom the infusion bag or bottle to the patient by gravity feed or by theuse of an infusion pump. The use of gravity feed infusion systems doesnot afford sufficient control over the rate of administration of theparenteral solution and, therefore, the use of an infusion pump ispreferred especially with solutions containing relatively highconcentrations of plasminogen activator or derivative thereof.Plasminogen activator. or a derivative thereof may also be orallyingested, topically applied, or inhaled as an aerosol.

The invention provides methods of administering a plasminogen activatorderivative to reduce oxidative injury, administering a plasminogenactivator derivative as an anti-inflammatory agent, applying aplasminogen activator derivative to reduce oxidant production by aneutrophil, and applying a plasminogen activator or derivative thereofto reduce oxidant production by a macrophage, monocyte, eosinophil, mastcell, or basophil. The production of oxidants by inflammatory cells maybe measured by cytochrome C reduction, chemiluminescence, orfluorescence detection. Neutrophil function may be assayed by a varietyof means (e.g., Bell et al., Br. Heart J. 63:82-87, 1990; Riesenberg etal., Br. Heart J. 73:14-19, 1995; Guidot et al., Am. J. Physiol.13:L2-L5, 1995). The role of oxidants in causing tissue injury isreviewed by Janssen et al. (Lab. Invest. 69:261-274, 1993) and Jaeschke(Proc. Soc. Exp. Biol. Med. 209:104-111, 1995).

In general, proteins (e.g., cytokines, monokines, receptors, proteases)may be measured by bioassay, ligand-receptor binding, immunoassay,and/or Western blotting. Histamine may be measured byfluorescence.(Shore et al., J. Pharmacol. Exp. Ther. 127:182-186, 1959).Nitric oxide may be measured by chemiluminescence (Hybertson et al.,Anal. Lett. 27:3081-3093, 1994). Proteases (e.g., elastase) may also bemeasured using a labeled peptide substrate (e.g., Barnett et al., J.Surg. Res. 63:6-10, 1996). Moreover, serotonin, arachidonate metabolites(e.g., prostaglandins, leukotrienes), cellular degranulation, andvascular permeability may be measured using assays well known in the art(see Handbook of Experimental Inmmunology).

Inflammatory diseases such as acute lung injury, acute respiratorydistress syndrome, arthritis, asthma, bronchitis, cystic fibrosis,hepatitis, inflammatory bowel disease, multiple sclerosis, reperfusioninjury (e.g., myocardial), nephritis, pancreatitis, psoriasis, arteryocclusion (e.g., retinal), stroke, systemic lupus erythematosus,transplantation, ultraviolet light induced injury, and/or vasculitis maybe treated using the invention. The inflammatory disease may be acute orchronic, and is preferably mediated by leukocytes (reviewed in Weissmannet al., Ann. N.Y. Acad. Sci. 389:11-24, 1982; Janoff, A., Annu. Rev.Med. 36:207-216, 1985; Hart et al., J. Rheumatol. 16:1184-1191, 1989;Doring, Am. J. Respir. Crit. Care Med. 150:S114-S117, 1994; Demling,Annu. Rev. Med. 46:193-202, 1995).

The invention also provides a method of screening structural variants ofplasminogen activator for their ability to act as anti-inflammatoryagents. Activity as an anti-inflammatory agent may be assayed by oxidantproduction by an inflammatory cell (e.g., neutrophil, macrophage,monocyte, eosinophil, mast cell, basophil); the carrageenan rat footpadmodel; and/or interleukin-1 induced pulmonary injury. In addition,structural variants of plasminogen activator may be screened forfibrinolytic activity and/or binding to a receptor for plasminogenactivator.

All books, articles, applications, and patents cited in thisspecification are incorporated herein by reference in their entirety.This includes the priority document U.S. Appln. No. 60/036,566 filedJan. 29, 1997. Such references are also cited as indicative of the skillin the art.

The following examples are meant to be illustrative of the presentinvention, however the practice of the invention is not limited orrestricted in any way by them.

EXAMPLE 1

Plasminogen Activator Inhibits Oxidant Production

The following example was published after the filing date of thepriority document as Stringer et al. (Inflammation 21:27-34, 1997).

Recovery and Purification of Human Neutrophils

Human neutrophils were isolated from the whole blood of a single,healthy, drug-free donor using a percoll density gradient (Polymorphprepfrom Nycomed, Oslo, Norway) (Ferrante and Thong, J. Immunol. Meth.36:109, 1994). Cells were then suspended inKrebs-Ringers-Phosphate-Dextrose (KRPD) buffer (serum-free), counted,and assessed for viability using trypan blue exclusion. Tissueplasminogen activator (tPA, alteplase from Genentech, South SanFrancisco, Calif.) was reconstituted following the manufacturer'sinstructions using sterile water for injection to produce a finalconcentration of 1 mg/ml. All experiments were performed at 37° C. andpH 7.4, under sterile conditions. Measurement of Neutrophil O₂ ⁻Generation by tPA

tPA was added to the neutrophil suspension in sufficient quantities toproduce final concentrations of 5, 20, 40, or 100 μg/ml. The effect ofL-arginine on neutrophil O₂ ⁻ generation was also evaluated becauseL-arginine is a precursor of nitric oxide (NO) and the standardformulation of tPA contains 700 mg L-arginine/20 mg tPA. L-arginine(Sigma Chemical Co., St. Louis, Mo.) concentrations of 175, 700, 1400,or 3500 μg/ml were evaluated that corresponded to the tPA concentrationsused above. Release of O₂ ⁻ by neutrophils (5×10⁶ cells/ml) stimulatedwith phorbol myristate acetate (PMA, 1.25 μg/ml) was determined during a30 min incubation in the absence or presence of each concentration oftPA or L-arginine. O₂ ⁻ generation was determined spectrophotometricallyby measuring superoxide dismutase (SOD) inhibitable horse heartferricytochrome C reduction (Babior et al., J. Clin. Invest. 52:741-744,1973; Fantone et al., Biochem. Biophys. Res. Comm. 113:506-512, 1983).Experiments were performed in triplicate.

PPACK Inhibition of tPA

D-Phe-Pro-Arg-chloromethyl ketone HCl (PPACK, Calbiochem, San Diego,Calif.), is an irreversible serine protease inhibitor, that inhibits theproteolytic activity of tPA in vitro (Lijnen et al., Thromb. Res.34:431-437, 1984). tPA was incubated in the presence of PPACK at varyingmolar ratios (PPACK:tPA: 5:1, 25:1, 100:1, or 1000:1) for 10 min afterwhich PPACK:tPA complexes or tPA alone (100 μg/ml) were incubated withplasminogen (375 μg/ml) for 5 hr in a cell incubator (5% CO₂ in air) at37° C. Subsequently, 50 μl of each of the incubated samples weresubjected to 7.5% acrylamide gel electrophoresis along with tPA (100μg/ml), plasminogen (375 μg/ml), and plasmin (1 U/ml). Each gel was runat 30V for 16 hr and protein bands were visualized by Coomasie bluestain. The effect of the PPACK:tPA complex on human neutrophil O₂ ⁻production was also examined. Briefly, the cell suspension (5×10⁶cells/ml) was divided into four groups: tPA (100 μg/ml); PPACK:tPA(5:1); PPACK (140 μM) alone, and PPACK vehicle (10 mM acetic acid).Cells (250 μl of 5×10⁶/ml) from each group were then plated into a96-well microtiter plate and incubated for 30 min at 37° C. in a cellincubator. Cells were then exposed to PMA (1.25 μg/ml) so that thefollowing conditions were met (in triplicate): tPA alone, tPA+PMA,PPACK:tPA alone, PPACK:tPA+PMA, PPACK alone, PPACK+PMA, PMA alone, PPACKvehicle, and cells alone. The plate was then incubated for an additional30 min at 37° C. in a cell incubator after which it was placed in aplate reader (Spectramax, Molecular Devices, Menlo Park, Calif.) and O₂⁻-production was measured as cytochrome C reduction (550 nm OD) everyfive min for 2 hr (Waud et al., Arch. Biochem. Biophys. 169:695-701,1975). The kinetic disposition of each treatment was compared.

Measurement O₂ ⁻ of Scavenging by tPA

The ability of tPA to scavenge O₂ ⁻ was determined by measuringreduction of cytochrome C during a 30 min incubation with purifiedxanthine oxidase (1.6 U/ml) and hypoxanthine in the presence or absenceof tPA (concentrations previously mentioned) (Waud et al., ibid.).Experiments were performed in triplicate.

Analyses of Data

The mean and standard error of the mean (±SEM) for data were determinedfor each experiment. Treatment groups were compared to each other and topositive and negative controls by analyses of variance and unpairedstudent's t tests. Concentration dependent effects were assessed bylinear regression followed by an F test for significance. A p value ofless than 0.05 was considered significant.

Effect of tPA on Neutrophil O₂ ⁻ Generation In Vitro

Adding increasing amounts of tPA significantly (p<0.025) andprogressively decreased O₂ ⁻ production by human neutrophils stimulatedby PMA in vitro (FIG. 1). In contrast, adding L-arginine, a component ofthe tPA formulation, did not decrease (p>0.05) O₂ ⁻ production byneutrophils stimulated with PMA (FIG. 2). Neither tPA nor L-argininealtered neutrophil O₂ ⁻-production by unstimulated neutrophils.

Effect of tPA Proteolytic Activity on O₂ ⁻ Production

tPA promoted conversion of plasminogen to plasmin. tPA mediatedconversion of plasminogen to plasmin was inhibited by PPACK in aconcentration dependent fashion. Based on these results, the mole:mole(PPACK:tPA) ratio used in the subsequent experiments was 5:1. In thesestudies, both tPA and PPACK-treated, proteolytically inactivated, tPAcomparably inhibited O₂ ⁻ production by neutrophils stimulated with PMA(FIG. 4). In addition, analysis of the kinetics of O₂ ⁻ productionshowed that both tPA and proteolytically inactivated tPA decreased theVmax of O₂ ⁻ production (i.e. rate of cytochrome reduction) similarly(FIG. 3).

Effect of tPA on O₂ ⁻ Generation by Xanthine Oxidase In Vitro

Adding tPA did not decrease O₂ ⁻ concentrations produced by xanthineoxidase (XO) in vitro (FIG. 4).

tPA significantly reduced O₂ ⁻ production by PMA stimulated humanneutrophils in vitro. The inhibitory effect of tPA was not dependent ontPA proteolytic activity, not related to L-arginine in its formulation,and not a consequence of its direct scavenging of O₂ ⁻. Theseobservations show that tPA has another action, inhibition of neutrophilO₂ ⁻ production, which may be used to reduce neutrophil O₂ ⁻ productionand prevent oxidative injury.

These results indicate that tPA acts directly on the neutrophil toreduce O₂ ⁻ production, independent of fibrinolytic activity. Theseobservations could have important clinical implications for optimizingthe efficacy of tPA in the management of myocardial infarction as wellas other inflammatory processes where a contribution by neutrophilderived O₂ ⁻ is likely. Indeed, the possibility that tPA might haveanti-inflammatory effects is supported by our related in vivo findingsshown below.

EXAMPLE 2

Plasminogen Activator's Anti-Inflammatory Effect in the CarrageenanInduced Rat Footpad Model

Carrageenan, a mucopolysaccharide derived from Irish sea moss, is aphlogistic agent that provokes a local antigenic inflammatory responsewhich is primarily attributed to neutrophil mediated injury and ishighly reproducible (Vinegar et al., Fed. Proc. 35:2447-2456, 1976;Vinegar et al., J. Pharmacol. Exp. Therap. 166:96-103, 1969; Vinegar etal., Eur. J. Rheumatol. Inflam; 1:204-211, 1978). This model has beenused extensively to evaluate the anti-inflammatory effects of such drugsas the non-steroidal anti-inflammatory drugs, corticosteriods, and morerecently superoxide dismutase (Winter and Flataker, J. Pharmacol. Exp.Therap. 150:165-171, 1965; Vinegar et al., Fed. Proc. 46:118-126, 1987;Ando et al., Biochim. Biophys. Acta 1073:374-379, 1991).

The following example was published after the filing date of thepriority document as Stringer et al. (Free Radicals Biol. Med.22:985-988, 1997).

The right hind foot volume of male Sprague-Dawley rats weighing between200-250 grams was determined using water-displacement prior tocarrageenan or carrageenan vehicle (saline) injection. Following initialbaseline (pretreatment) foot volume determinations, the rats werelightly anesthetized using methoxyflurane (Pittman-Moore, Mundelcin,Ill.) and 0.10 ml of 1.5% (w/v) carrageenan (Sigma Chemical Co., St.Louis, Mo.) in sterile normal saline, or saline (0.10 ml, sterile normalsaline) was injected into the plantar tissue of the right hind paw.Volume of the injected paw was measured 30 min and then every hour for 6hr thereafter.

Treatments

Both SK and tPA were reconstituted according to manufacturers'instructions. Baseline footpad volume measurements were made immediatelyprior to carrageenan or saline administration.

Tissue plasminogen activator (tPA, alteplase from Genentech, South SanFrancisco, Calif.): Three different doses of 3, 6, and 12 mg/Kg bodyweight tPA were evaluated. Half of each dose was given intraperitoneally(i.p.) 10 min prior to footpad carrageenan injection. The second half ofthe dose was administered 2.5 hr after the first half of the tPA dose.This treatment regimen was considered necessary to account for the shorthalf-life of tPA which is approximately 5 min (Tbbe et al., Am. J.Cardiol. 64:448-453, 1989).

L-arginine (Sigma Chemical Co., St. Louis, Mo.): The formulation of tPA(from Genentech) contains L-arginine to enhance solubility., In so faras L-arginine is a precursor of nitric oxide (NO), the effect ofL-arginine on rat footpad inflammation was evaluated. Doses ofL-arginine (0.1 1, 0.22, 0.44 g/Kg body weight, i.p.) utilizedcorrespond to those contained in the tPA doses.

Streptokinase (SK, KABIKINASE® from Kabi-Vitrum, Sweden): Rats receivedone of three single SK doses (10,000, 20,000, or 40,000 U/Kg bodyweight, i.p.) 10 min prior to the carrageenan footpad injection.

Histological Examination

Upon completion of the experiments, the animals were sacrificed andtheir paw removed, fixed in formalin, sectioned, and stained withhematoxylin and eosin. Sections were examined and assessed forneutrophil infiltration by an individual unaware of the treatmentschemes. Neutrophils from six high powered fields (40×) of arepresentative slide from the highest dose of each PA treatment and thecarrageenan and vehicle controls were counted and averaged.

Data Analysis

Calculation of the edema index: An edema index was calculated for eachfootpad as a measure of inflammation. This was determined by subtractingthe weight of the water-filled tube following insertion of the paw ateach time point from the weight of the water-filled tube. Edema inducesa greater displacement of water. The time zero (pretreatment) footvolume was then subtracted from each time point so that changes involume reflected those associated with edema. The mean (±SEM) edemaindex for each time point for each group was determined. The edemaindexes for each PA or L-arginine group were compared to carrageenancontrol group at each time point using a Mann-Whitney two sample test.In all cases, a p value less than 0.05 was considered significant.

Histological examination: The mean (±SEM) neutrophil count per highpowered field (HPF) was determined for each treatment and compared tothe carrageenan control using analysis of variance (ANOVA).

Carrageenan induced edema when injected into the rat footpad (FIGS.5-6). tPA reduced edema in a dose-dependent manner (FIG. 5). At a doseof 12 mg/Kg body weight, tPA reduced edema at all time points (p<0.05)while 6 mg/body weight reduced edema beginning at the two hour timepoint (p<0.05); an effect that occurred prior to the second dose of tPA.The two highest doses of SK, 20,000 and 40,000 U/Kg body weight,enhanced edema at the latter time points (≦5 hr) (FIG. 6). By contrast,L-arginine, one of the constituents of the tPA formulation, had nosignificant effect on edema at any time.

Histological examination of the footpads revealed no significantdifferences in the number of neutrophils (mean+SEM) between thetreatment groups and carrageenan control (carrageenan control: 30.7±0.65cells/HPF; tPA: 35.0±12.6 cells/HPF; SK: 41.2±16.9 cells/HPF). Notably,the vehicle control footpads had no neutrophil infiltration.

This study shows that carrageenan induced inflammation can be modulatedby plasminogen activator. The effect was selective in that tPA inhibitededema development while SK enhanced it. Mechanisms by which drugs caninfluence carrageenan induced footpad inflammation and edema includeinhibition of neutrophil infiltration into the footpad, inflammatorymediator release, including neutrophil generated O₂ ⁻, and/or vascularpermeability. The first possibility is unlikely since there was nodifference between the plasminogen activators in regard to the magnitudeof neutrophil infiltration into the footpad. Generation of O₂ ⁻ exertsimportant proinflammatory effects, including deesterification ofphospholipids resulting in increased vascular permeability like thatobserved in ischemia-reperfusion injury (Deby et al., Biochem.Pharmacol. 39:399-405, 1990). tPA significantly reduces O₂ ⁻ productionby neutrophils is likely to contribute to the observed anti-inflammatoryeffect of tPA. The failure of L-arginine to affect edema assures thatL-arginine, an excipient in the tPA formulation, does not contribute tothe anti-inflammatory effect of tPA. This is consistent with ourprevious observation that L-arginine also does not alter neutrophil O₂ ⁻production in vitro.

Streptokinase, in contrast to tPA, enhanced inflammation as reflected inthe increase in edema index at the later time points. Although it is notknown whether plasminogen activators alter vascular permeabilitydirectly or indirectly via altercation of inflammatory mediator actionson endothelium, plasminogen activators have been shown to bind toendothelial cell surfaces (Hajjar et al., J. Clin. Invest. 80:1712-1719,1987). Thus, it may be speculated that the pro-inflammatory effect of SKis due, at least in part, a direct effect on blood vessels since SK hasno effect on neutrophil O₂ ⁻ production. This notion is supported by theobservation that SK has a vasoactive effect that is observed clinically,wherein most myocardial infarction patients treated with SK experiencesome degree of hypotension (a occurrence that is not observed inpatients treated with tPA). Such a vasodilatory action of SK maycontribute to the enhancement of edema.

EXAMPLE 3

Plasminogen Activator's Anti-Inflammatory Effect in the IL-1 InducedPulmonary Injury Model

Tissue plasminogen activator (tPA, alteplase from Genentech, South SanFrancisco, Calif.) was reconstituted according to the manufacturer'sinstructions. The total dose was 12 mg/Kg body weight givenintraperitoneally (i.p.); 6 mg/Kg as administered 10 min before IL-1 and6 mg/Kg was given 2.5 hours later. This regimen was chosen because ofthe short half-life of tPA (Tbbe et al., ibid.) and from the doseresponse study of Example 2. In addition, this dose of tPA does notincrease the activated partial thromboplastin time (aPTT) in rats(Example 2; Korninger et al., Thromb. Haemost. 46:561-565, 198 1). Tocontrol for possible effects of L-arginine contained in the formulationused, a corresponding dose of L-arginine (440 mg/Kg body weight, i.p.)(Sigma Chemical Co., St. Louis, Mo.) was administered similarly.

Determination of tPA Concentration in the Lung

To determine the effect of systemic administration of tPA on lung tPAconcentrations, six male Sprague-Dawley rats (300-400 gm) were given tPAas described above and then, five hours later, the lower left lobe ofthe lung was removed following euthanasia with methoxyflurane. Sampleswere stored at −80° C. until assay. Subsequently, samples were thawedand homogenized with ice-cold homogenization buffer (20 mMHEPES/glycerol buffer, pH 7.5), containing protease inhibitors (2 mMEDTA, 2 mM EGTA, 5 μg/ml aprotinin, 10 μM leupeptin, 1 mM PMSF) andcentrifuged at 15,000 G for 45 min. After the protein concentration ofeach supernatant was determined (Lowry et al., J. Biol. Chem.193:265-275, 1951), aliquots containing 100 μg protein were subjected to7.5% polyacrylamide gel electrophoresis and transferred tonitrocellulose (Towbin et al., Proc. Natl. Acad. Sci. USA 76:4350-4354,1979). These membranes were blocked with 3% skim milk in TNS buffer (15mM Tris, pH 7.4, 150 mM NaCl, 0.1% Tween-20) overnight and thenincubated with an antibody specific for tissue plasminogen activator(1:50 dilution of an anti-tPA sheep polyclonal antibody, affinitypurified IgG) (Enzyme Research, South Bend, Ind.) for 60 min at 25° C.Blots were then rinsed five times for 5 min each with wash buffer (3%skim milk in TNS) and incubated with a secondary polyclonal antibody(1:10,000 dilution of rabbit anti-sheep horseradish peroxidase) (JacksonImmunoResearch, West Grove, Pa.) for 30 min at 25° C. Following fiverinses (5 min each) with wash buffer, immunoblots were visualized byapplication of enhanced chemiluminescence (ECL) Western blottingreagents (Pierce, Rockford, Ill.) and exposure to autoradiographic film.Immunolabeled tPA was identified by comparison to a known concentrationof tPA (1 μM) run on the same gel.

Interleukin-1 Induced Acute Lung Injury

Ten minutes before intratracheal instillation of IL-1 (50 ng/0.5 ml ofrhIL-1α, R&D Systems, Minneapolis, Minn.) or vehicle (0.5 ml sterilesaline), tPA or L-arginine was administered to male (300-400 gm)Sprague-Dawley rats (Leff et al., Am. J. Physiol., 265:L501-L506, 1993;Leff et al., Am. J. Physiol. 266:L2-L8, 1994). The effect of L-argininewas evaluated since L-arginine, a precursor of nitric oxide synthesis invivo, is contained in the tPA formulation that was used.

After tPA or L-arginine administration, and anesthesia withmethoxyflurane (Pitman-Moore, Mundelein, Ill.), a 1 cm neck incision wasmade and the trachea was exposed by blunt dissection. A 25 gaugeangiocath was inserted through the tracheal wall and the Teflon catheteradvanced without the needle into the trachea. Saline (0.5 ml) or IL-1(50 ng) in saline (0.5 ml) was administered followed by two 3 ml puffsof air to ensure good distal delivery of the cytokine (Leff et al.,ibid.; Koh et al., J. Appl. Physiol. 79:472-478, 1995). Soft tissue wasreopposed and the neck incision sutured with three interrupted 3-0 silksutures. Five hours after IL-1α administration, lung leak, lungmyeloperoxidase (MPO) activity, and lung lavage neutrophil counts weredetermined (Leffet al., ibid.; Krawisz et al., Gastroenterology87:1344-1350, 1984).

Determination of Lung Leak and MPO Activity

Four and one-half hours after intratracheal instillation of saline orIL-1, rats were anesthetized with a mixture of ketamine (90 mg/Kg bodyweight) and xylazine (5 mg/Kg body weight) intraperitoneally and¹²⁵I-BSA (1.0 μCi in 0.5 ml) was administered intravenously. Twenty-fiveminutes thereafter, rats were ventilated using a Harvard small animalrespirator during laparotomy, thoracotomy, and right ventricularinjection of heparin (200 U in 0.2 ml). Right ventricular blood sampleswere obtained, lungs were perfused blood free with PBS and excised.Radioactivity in right lungs and blood samples were measured using agamma counter. Lung leak index was estimated as counts per minute (cpm)of ¹²⁵I in the lung divided by cpm in 1.0 ml of blood. Left lungs wereassayed for MPO activity using o-dianiside as substrate. Six rats wereutilized in the saline group (control), ten rats in the IL-1 group, andsix rats in the tPA and IL-1 group.

Determination of Lung Lavage Neutrophils

Five hours after tPA administration and instillation of saline or IL-1as described, rats were anesthetized using ketamine (90 mg/Kg bodyweight) and xylazine (7 mg/Kg body weight) intraperitoneally. Thetrachea was cannulated with an indwelling 16 gauge stub adaptor tube,and then saline (two×3.0 ml) was injected slowly and withdrawn (tolavage lungs). Recovered lavage fluid was centrifuged (250 G for 5 min)and the cell pellet resuspended in 1.0 ml of lavage supernatant; redblood cells were lysed using hypotonic saline. Total leukocytes werecounted using a Coulter counter and cytospin preparations of the cellswere Wright-Giemsa stained to determine the percentage and total numberof neutrophils. Ten rats were utilized in the IL-1 alone and tPA+IL-1experiments, while six rats were in the saline group (control).

Data Analysis

Data were analyzed using a one-way analysis of variance with aStudent-Newman-Keuls test of multiple comparisons. A p value of lessthan 0.05 was accepted as being statistically significant.

Rats that were treated with tPA (12 mg/Kg body weight, i.p.) hadincreased lung tPA levels (measured at 5 hours) compared to untreatedrats. Rats treated with tPA (12 mg/Kg body weight, i.p.) had anapproximately 80% reduction in lung leak compared to untreated ratsgiven IL-1 intratracheally (FIG. 7). Lung leak in rats given L-arginine(440 mg/Kg body weight, i.p.) along with IL-1 was not different fromlung leak in rats given IL-1 (FIG. 9). In contrast, rats given both tPAand IL-1 had the same number of lavage neutrophils and lung MPOactivities as untreated rats given IL-1 intratracheally (Table 1).Consistent with the in vivo data of Example 2, tPA failed to abrogateneutrophil migration induced by an inflammatory stimulus in vivo (Table1).

TABLE 1 Effect of tPA on lung lavage neutrophils (PMNs) and lung MPOactivity in rats given IL-1 intratracheally. Lung MPO in whole Lunglavage PMNs lavage PMNs lung (U/gm left Treatment (% total cells) (total#, millions) lung) control*  3 ± 1 0.003 ± 0.001  0.6 ± 0.2 IL-1  95 ±1⁺  2.9 ± 0.4⁺  11.2 ± 2.9⁺ tPA + IL-1   95 ± 1⁺{circumflex over ( )}  2.7 ± 0.4⁺{circumflex over ( )}   11.1 ± 1.6⁺{circumflex over ( )}*mean ± SEM of six determinations. ⁺value significantly different (p <0.05) from control value; mean ± SEM of ten determinations. {circumflexover ( )}value not significantly different (p > 0.05) from valueobtained for rats given IL-1 alone; mean ± SEM of six determinations.

Lung leak, lung myeloperoxidase (MPO) activity, and lung lavageneutrophil counts were increased in rats given IL-1 intratracheallycompared to control rats that were given saline intratracheally. GivingtPA (12 mg/Kg body weight) intraperitoneally increased lung tPAconcentration and reduced acute lung leak in rats given IL-1intratracheally (p<0.01). Lung leak index for sham treatment was0.040±0.001 (n=6), IL-1 treatment was 0.10±0.01 (n=10), and tPA+IL-1treatment was 0.050±0.002 (n=6). In contrast, administering tPA did notchange the IL-1 induced increases in lavage neutrophils (sham treatmentwas 3±1×10³ cells, IL-1 treatment was 2.9±0.4×10⁶ cells, and tPA+IL-1treatment was 2.7±0.4×10⁶ cells) or lung MPO activity (sham treatmentwas 0.6±0.2 U/gm lung, IL-1 treatment was 11.2±2.9 U/gm lung, andtPA+IL-1 was 11.1±1.6 U/gm lung). We have demonstrated that IL-1 inducedneutrophil-dependent lung injury can be modulated by tPA. Administrationof intraperitoneal tPA increases lung tissue tPA levels and decreasesacute lung injury, without reducing lung neutrophil infiltration in ratsgiven IL-1α intratracheally. As the systemic administration of tPAresulted in a measurable increase in tPA concentration in the lung,inhibition of lung injury is probably due to an inhibitory effect of tPAon neutrophil O₂ ⁻ production.

EXAMPLE 4

Plasminogen Activator's Anti-Inflammatory Effect on Macrophages

Chemiluminescence was used to measure the oxidative burst of ratalveolar macrophages (NR 8383 cells). Oxidant production was determinedby luminol chemiluminescence measured using a luminometer (Lumistar, BMGLab Technologies Inc., Durham, N.C.) (Archer et al. J. Appl. Physiol.67:1912-21, 1989). Experiments were conducted in an opaque 96-well plateat 37° C. Suspensions of macrophages (100 μl of 5 million cells/ml) wereplated in the presence or absence of tPA (100 μg/ml) 60 min prior toexposure to an activator (PMA, zymosan, or opsonized zymosan). Prior tothe addition of activator, 200 μl of buffered luminol solution (0.1 μM)containing horseradish peroxidase (0.5 mg/ml) was added to each well andchemiluminescent light emission was determined (baseline was measured attime 0). Following addition of an activator, chemiluminescent lightemission was measured every 10 min for two hours. The experiments wereperformed in triplicate. The assay has a detection limit ofapproximately 100 nM hydrogen peroxide.

Addition of an activator resulted in an increase in oxidant productionby the macrophages (FIG. 8). tPA reduced activator induced oxidantproduction, demonstrating that the ability of tPA to inhibit oxidantproduction is not selective for neutrophils but, instead extends todifferent types of leukocytes.

EXAMPLE 5

Evaluation of the Effect of Tissue Plasminogen Activator (tPA) onNeutrophil Apoptosis

Neutrophils were isolated from the whole blood of a single, healthy,medication-free individual using venipuncture and methods previouslydescribed (Stringer et al., Inflammation 21:27-34, 1997). Cells (1×10⁶cells/ml) were suspended in Krebs-Ringers-Phosphate-Dextrose (KRPD)buffer and equally divided between two tubes. To one tube, tissueplasminogen activator (tPA) was added to produce a final concentrationof 100 μg/ml. Cell suspension (200 μl) was placed in each well of a96-well microtiter plate and the plate was incubated (37° C., 5% CO₂)for 30 min. Following the incubation, phorbol myristate acetate (PMA,1.25 μg/ml) or formyl-methionyl-leucyl-phenylalanine (fMLP, 5 μM) wasadded to wells so that the following conditions were met: cells alone,tPA alone, tPA+PMA, tPA+fMLP, PMA alone, or fMLP alone. The plate wasthen incubated again (37° C., 5% CO₂) for 30 min.

The percent apoptotic cells was determined at time 0 (immediatelyfollowing incubation), then at 4, 8, 12, 16, 20, and 24 hr. At each timepoint, cells (25 μl) were removed from each well of the microtiter plateand placed into a glass tube with 1 μl of ethidium bromide/acridineorange (4 μg/ml each). Cells (10 μl) were then placed on a microscopeslide with a cover slip. Cells were viewed under a microscope (100×)equipped with a fluorscein filter. For each assessment, cells (n=100)were counted and “scored” as either “live apoptotic”, “live normal”,“dead apoptotic”, or “dead normal” (Duke et al. In: Current Protocols inImmunology, edited by Coligan et al., John Wiley & Sons, New York, pp.3.17.1-3.17.33, 1995). The percent apoptotic cells was determined byadding the number of live and dead apoptotic cells.

tPA alone significantly reduced the rate of apoptosis and the percentapoptotic cells at 24 hours (FIG. 9). The rate and magnitude ofapoptosis was significantly enhanced by PMA while fMLP had no effect.The addition of tPA significantly slowed the rate and reduced themagnitude of apoptosis in PMA-treated cells, and reduced the rate andmagnitude of apoptosis in fMLP-treated cells.

While the present invention has been described in connection with whatis presently considered to be practical and preferred embodiments, it isunderstood that the present invention is not to be limited or restrictedto the disclosed embodiments but, on the contrary, is intended to covervarious modifications and equivalent arrangements included within thescope of the appended claims.

Thus, it is to be understood that variations in the described inventionwill be obvious to those skilled in the art without departing from thenovel and non-obvious aspects of the present invention, and suchvariations are intended to come within the scope of the claims below.

We claim:
 1. A method of treating a subject with an inflammatory lungdisease or condition comprising: administering to a subject with aninflammatory lung disease or condition a therapeutically effectiveamount of a tissue plasminogen activator protein havinganti-inflammatory activity, wherein a therapeutically effective amountis an amount of said tissue plasminogen activator (tPA) proteinsufficient to reduce inflammation or inflammation-dependent lung damagein said subject.
 2. The method of claim 1 wherein said inflammatory lungdisease is selected from the group consisting of acute lung injury,acute respiratory distress syndrome, asthma, bronchitis, and cysticfibrosis.
 3. The method of 2 where the inflammatory lung disease isacute respiratory distress syndrome (ARDS).
 4. The method of claim 1wherein said tissue plasminogen activator protein is administered viaoral, topical, inhalation or parental administration.
 5. The method ofclaim 4 wherein said tissue plasminogen activator protein isadministered via inhalation.
 6. The method of claim 5 wherein saidtissue plasminogen activator protein is administered via intravascularinfusion.
 7. The method of claim 6 where said tissue plasminogenactivator protein is administered by use of an aerosol composition.